CN111217917A

CN111217917A - Novel coronavirus SARS-CoV-2 vaccine and preparation method thereof

Info

Publication number: CN111217917A
Application number: CN202010120290.9A
Authority: CN
Inventors: 李军强; 司伟雪; 崔海燕; 李荩; 巢守柏; 朱涛
Original assignee: CanSino Biologics Inc
Current assignee: CanSino Biologics Inc
Priority date: 2020-02-26
Filing date: 2020-02-26
Publication date: 2020-06-02
Anticipated expiration: 2040-02-26
Also published as: CN111217917B

Abstract

The invention relates to a preparation method of a vaccine for treating and/or preventing a novel coronavirus (SARS-CoV-2) infection or a novel coronavirus disease (COVID-19), wherein a core antigen of the vaccine comprises RBD fusion protein of the novel coronavirus (SARS-CoV-2), and the form of the vaccine comprises RBD fusion protein subunit vaccine, RBD fusion protein mRNA vaccine or RBD fusion protein adenovirus vector vaccine. The vaccine can generate immune response for treating and/or preventing infection of novel coronavirus (SARS-CoV-2) after immunizing organism, and can be used for treating and/or preventing novel coronavirus disease (COVID-19). The invention also relates to RBD fusion gene, RBD fusion protein, carrier, cell, preparation method, treatment method or pharmaceutical application of the novel coronavirus (SARS-CoV-2).

Description

Novel coronavirus SARS-CoV-2 vaccine and preparation method thereof

Technical Field

The invention belongs to the technical field of vaccines, and particularly relates to a RBD fusion gene of a novel coronavirus (SARS-CoV-2) which is designed into an RBD fusion protein subunit vaccine, an RBD fusion protein mRNA vaccine or an RBD fusion protein adenovirus vector vaccine, wherein the vaccine can generate an immune response for preventing the novel coronavirus (SARS-CoV-2) after immunizing an organism and is used for preventing a novel coronavirus disease (COVID-19).

Background

coronaviruses are nonsegmented, single-stranded, positive-strand RNA viruses belonging to the group of the nested (Nidovirales) coronaviruses (Coronaviride) the orthocoronaviridae (Orthocoronavirinae), which are divided into α, beta, gamma and delta, depending on the serotype and genomic characteristics, to date, a total of 7 coronaviruses infect humans, including 229E and NL63 of the β genus, OC43 and HKU1 of the beta genus, middle east respiratory syndrome-associated coronavirus (MERSR-CoV), severe acute respiratory syndrome-associated coronavirus (SARSr-CoV) and novel coronavirus (SARS-CoV-2).

The coronavirus is a positive-strand single-stranded RNA virus with an outer mantle, the diameter of the coronavirus is about 80-120 nm, the genome of the coronavirus is 27-32 Kb, the genetic material of the coronavirus is the largest of all RNA viruses, and the coronavirus has the important structural characteristics of the positive-strand RNA: namely, the 5 'end of the RNA chain is provided with a methylated cap, and the 3' end is provided with a polyA tail structure. The structure is very similar to eukaryotic mRNA and is an important structural basis for the genome RNA to play the role of a translation template, so that the transcription process of RNA-DNA-RNA is omitted, and the characteristic makes the genome RNA extremely easy to mutate. Coronavirus can infect vertebrates such as human, mouse, pig, cat, dog, bird, etc. Coronaviruses have envelopes, spinous processes exist on the envelopes, the whole virus is like coronas, and the spinous processes of different coronaviruses have obvious differences. Tubular inclusions are sometimes visible in coronavirus infected cells.

The S protein is the most important surface protein of coronavirus, and is related to the infectivity of virus. The S protein contains two subunits: s1 and S2, wherein S1 comprises mainly a Receptor Binding Domain (RBD) responsible for recognizing cellular receptors; s2 contains essential elements required for the membrane fusion process. The S protein has the functions of combining virus and host cell membrane receptors and fusing membranes; the S protein determines the host range and specificity of the virus; the S protein can realize transmission among different hosts through gene recombination or mutation of a receptor binding Region (RBD), and leads to higher lethality rate; the S protein can generate neutralizing antibodies, and therefore, the S protein is an important candidate antigen for vaccine design.

Although S protein is the first antigen for coronavirus vaccine development, it is related to SARS-CoV vaccine research, and the results show that S protein induced antibody can induce Antibody Dependent Enhancement (ADE); at present, there are few reports on the development of novel coronavirus vaccines, but considering that the homology between the novel coronavirus and SARS is high, it is an important issue to consider whether a vaccine designed against the S protein of the novel coronavirus can cause ADE at the beginning of vaccine design.

The invention designs fusion protein based on RBD, and the antibody produced by the fusion protein can prevent the novel coronavirus from being recognized by host cells. The function of RBD in the novel coronavirus S protein is clear and is responsible for recognizing ACE (angiotensin converting enzyme) region of a receptor cell, so that the function of an antibody generated aiming at the RBD is clear, and the Antibody Dependence Enhancement (ADE) generated by an induced organism is avoided.

Disclosure of Invention

The invention provides a novel RBD fusion protein of coronavirus (SARS-CoV-2), the structure form of the fusion protein is RBD-CTB or RBD-CRM197(A), wherein RBD is a receptor binding region, and the generated antibody can interfere the recognition of virus and host cells, thereby preventing the virus from entering the host cells; CTB is a B subunit of cholera toxin, is helpful for transferring antigen into cells, induces and generates TH1 and TH2 immune pathways, and has the effect of a mucosal immune adjuvant; CRM197(A) is an A region of CRM197, and the region has a plurality of CTL epitopes, and fusion protein formed by using CRM197(A) as a monomer can generate stronger cellular immunity.

Preferably, the two monomers of the fusion protein are positioned in a way that they can be substituted for each other, and either monomer can be placed in front of the other without affecting the effect of each monomer, i.e., RBD-CTB, CTB-RBD or RBD-CRM197(A), CRM197(A) -RBD.

Preferably, the RBD fusion protein further comprises a signal peptide, more preferably a tPA signal peptide, and more preferably the gene sequence thereof is shown in SEQ ID NO. 5.

Preferably, the fusion protein further comprises Linker arms (linkers), more preferably G4S, between different protein monomers, and the main function of the Linker arms is to ensure that each protein maintains its own conformation.

The invention provides an RBD fusion protein, which comprises a molecular adjuvant, namely CPG or TLR (TLR agonist) besides CTB or CRM197(A) serving as the fusion protein of the RBD.

Preferably, the molecular adjuvant-containing RBD fusion protein further comprises a signal peptide, more preferably the signal peptide is tPA, and more preferably the gene sequence is shown as SEQ ID NO. 5.

Preferably, the RBD fusion protein containing molecular adjuvant further comprises a Linker (Linker) between different protein monomers, wherein the Linker is commonly used as G4S, and the main function of the Linker is to ensure that each protein maintains its own conformation.

In one embodiment of the invention, based on the fusion protein, the designed vaccine is a recombinant protein subunit vaccine, an RBD fusion gene is connected with pET9a, an expression plasmid is constructed, and is transferred into escherichia coli BL21, and a target antigen is purified, so that the subunit vaccine is obtained.

In one embodiment of the invention, based on the fusion protein, the designed vaccine is in the form of an adenovirus vector vaccine, and the RBD fusion gene is inserted into an adenovirus vector to form the adenovirus vector vaccine.

Preferably, the adenovirus containing the RBD fusion protein antigen is obtained by cotransfection of an adenovirus backbone plasmid and a shuttle plasmid containing an antigen gene. The adenovirus backbone plasmid is selected from: pAdEasy-1, pAdEasy-2, pBHG11, pBHG-fiber5 or pBHG-fiber 35. The packaging system comprises: AdEasy or adamax.

Preferably, the method for constructing a shuttle plasmid containing an antigen gene comprises: synthesizing an RBD fusion gene sequence, designing and introducing enzyme cutting sites which are the same as those of the shuttle plasmid at the two ends of the antigen gene sequence, carrying out double enzyme cutting on the target antigen and the shuttle plasmid, recovering nucleotide fragments, and connecting the antigen sequence and the plasmid by T4 DNA ligase.

In one embodiment of the invention, the vaccine designed based on the fusion protein is an mRNA vaccine.

preferably, a plasmid containing a fusion gene is constructed, escherichia coli DH5 α is transformed, a positive single spot is selected, a liquid culture medium is inoculated, a strain is expanded, bacteria are cultured in a shake flask or a fermentation tank, bacteria are centrifugally recovered, the plasmid is extracted, single enzyme digestion is carried out to linearize the plasmid, and the plasmid is used as a template to synthesize mRNA.

Preferably, the synthesized mRNA is preceded by a cap, and is methylated, and the tail is added with polyA.

The invention provides a preparation method of a vaccine for treating and/or preventing a novel coronavirus (SARS-CoV-2) infection or a novel coronavirus disease (COVID-19), which comprises constructing a fusion protein containing the novel coronavirus (SARS-CoV-2), wherein the structural form of the fusion protein is antigen-CTB-TLR of the fusion protein or antigen-CRM 197-TLR of the fusion protein.

Preferably, the antigen of the fusion protein is any antigen or antigen fragment capable of producing protection on the novel coronavirus, including S antigen, S1 antigen, E antigen, M antigen or N antigen, and combinations of different antigens, such as S + N, S + E + N, etc.

Preferably, the antigen of the fusion protein is the RBD of S1.

Preferably, the vaccine for treating and/or preventing the infection of the novel coronavirus (SARS-CoV-2) or the novel coronavirus disease (COVID-19) is in the form of a subunit vaccine obtained by purifying after expressing the fusion gene. Or the fusion protein gene is inserted into an adenovirus vector to obtain the adenovirus vector vaccine. Or the fusion protein gene is used as a template, and the mRNA vaccine is obtained by amplification.

The invention takes RBD as a target antigen, and the generated antibody can prevent a novel coronavirus (SARS-CoV-2) from entering a host cell; RBDs are Receptor Binding Domains (RBDs) responsible for recognizing cellular receptors, and antibodies directed against RBDs can be effective in avoiding potential ADE. The RBD amino acid fragment has smaller molecular weight, and the RBD is used as an antigen to difficultly generate higher antibody titer. More importantly, the fusion protein constructed by the invention is added with an adjuvant, so that the immunogenicity of the antigen is greatly improved.

the product, medicament, vaccine of the invention may further comprise a pharmaceutically acceptable excipient, which may be a carrier, diluent, adjuvant or nucleotide sequence encoding adjuvant, suspending agent, transfection facilitating agent, etc. the nucleotide sequence encoding adjuvant is a nucleotide sequence encoding at least one of GM-CSF, IL-17, IFNg, IL-15, IL-21, anti-PD 1/2, lactoferrin, protamine, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF- α, INF- γ, Lymphotoxin- α, hGH-1, hGH-MCP-1, MIP-1 a, MIP-1P, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD 5, GlyCAM-1, MadCAM-1, VLA-581, TRAIL-8, RANTES, TRAIL-6, TRACKET-6, TRACKIRC-6, TRACKET-5, TRACKROCK-1, TRACKET-5, TRACKET-1, TRACKET-5, TRACKET-3, TRACKET-5, TRACKET-1, TRACKET-5, TRACKET-3, TRACKET-III, TRACKET-5, TRACKET-III-5, TRACKET-3, TRACKET-III-3, TRACKET-5.

In some more specific embodiments, the first aspect of the present invention relates to an RBD fusion protein comprising:

(1) RBD amino acid fragments of novel coronavirus SARS-CoV-2;

(2) a Linker is connected with the arm;

(3) CTB or CRM197(A) amino acid fragment.

Preferably, the fusion protein as described above,

(1) the sequence of the RBD amino acid fragment of the novel coronavirus SARS-CoV-2 is shown as SEQ ID NO: as shown in fig. 6, and/or,

(2) the sequence of the connecting arm Linker is shown as SEQ ID NO: 7, and/or,

(3) the CTB amino acid fragment has a sequence shown in SEQ ID NO: as shown in fig. 8, and/or,

(4) the CRM197(A) amino acid fragment has a sequence shown in SEQ ID NO: shown at 9.

More preferably, the fusion protein as described above, further comprising: a signal peptide and/or a molecular adjuvant; preferably, the signal peptide is connected to the N end of the fusion protein, and the molecular adjuvant is connected to the C end of the fusion protein; more preferably, the signal peptide is represented by the sequence shown in SEQ ID NO: and 7, connecting a Linker arm Linker shown in the figure and the fusion protein.

More preferably, the fusion protein is characterized in that,

(1) the signal peptide is tPA signal peptide, and/or,

(2) the molecular adjuvant is CPG or TLR.

The second aspect of the present invention relates to a fusion gene characterized in that,

(1) encoding a fusion protein as described in any one of the above;

alternatively, the first and second electrodes may be,

(2) it includes: an RBD coding gene sequence of a novel coronavirus SARS-CoV-2, a coding gene sequence of a Linker arm Linker, and a coding gene sequence of CTB or CRM197 (A).

The third aspect of the present invention relates to a vector comprising the fusion gene as described above.

A fourth aspect of the invention relates to a host expressing a fusion protein as described above, comprising a fusion gene as described above and/or comprising a vector as described above.

Preferably, the host may be a cell or a bacterium.

The fifth aspect of the present invention relates to a vaccine for treating and/or preventing a novel coronavirus SARS-CoV-2 infection or a novel coronavirus disease COVID-19, which comprises or is prepared from the above-mentioned fusion protein, the above-mentioned fusion gene, the above-mentioned vector, or the above-mentioned host.

Preferably, the vaccine as described above, wherein said vaccine is in the form of a recombinant protein subunit vaccine, a recombinant protein mRNA vaccine or a recombinant protein adenoviral vector vaccine.

The sixth aspect of the present invention relates to a method for preparing a vaccine for preventing and/or treating a novel coronavirus SARS-CoV-2 infection or a coronavirus disease COVID-19, comprising the steps of:

(1) the fusion gene as described above was constructed,

(2) expressing the fusion protein according to any one of the above,

(3) purifying to obtain subunit vaccine;

alternatively, the first and second electrodes may be,

(1) the fusion gene as described above was constructed,

(2) inserting the fusion gene into an adenovirus vector,

(3) preparing an adenovirus vector vaccine;

alternatively, the first and second electrodes may be,

(1) using the fusion gene as described above as a template,

(2) and amplifying to obtain the mRNA vaccine.

The seventh aspect of the present invention relates to the use of the fusion protein as described above, the fusion gene as described above, the vector as described above, the host as described above and/or the vaccine as described above in the preparation of a medicament or product for preventing and/or treating a novel coronavirus SARS-CoV-2 infection and/or a novel coronavirus disease COVID-19.

An eighth aspect of the present invention relates to a method for preventing and/or treating a novel coronavirus SARS-CoV-2 infection and/or a novel coronavirus disease covi-19, comprising administering to an individual a vaccine according to any one of the fusion protein as described above, the fusion gene as described above, the vector as described above, the host as described above and/or the vaccine as described above.

A ninth aspect of the invention relates to a method of inducing a neutralizing antigen-specific immune response in an individual, characterized in that a fusion protein as described above, a fusion gene as described above, a vector as described above, a host as described above and/or a vaccine as described in any of the above is administered to the individual.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 coronavirus plan view

FIG. 2 is a graph showing the results of double digestion of RBD-Linker-CTB fusion gene-T vector

FIG. 3 shows the result of induced expression of RBD-linker-CTB fusion gene

FIG. 4 flow chart of RBD fusion protein adenovirus vector vaccine packaging

FIG. 5 purity analysis (HPLC) of the RBD-Linker-CTB fusion protein adenoviral vector vaccine

FIG. 6RBD-Linker-CTB fusion protein immunogenicity (ELISA method)

FIG. 7 Effect of molecular adjuvants (TLR) on the immunogenicity of RBD-Linker-CTB fusion proteins (ELISA method)

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 construction of RBD-Linker-CTB fusion Gene of novel coronavirus (SARS-CoV-2)

The structure and coat protein composition of the novel coronavirus (SARS-CoV-2) are shown in FIG. 1. The S protein is the most important surface protein of coronaviruses, and contains two subunits: s1 and S2, in which the Receptor Binding Domain (RBD) is distributed in the S1 region and is responsible for recognizing cellular receptors; the S protein can be transmitted among different hosts through gene recombination or mutation of a receptor binding Region (RBD), and causes higher lethality. More importantly, coronaviruses recognize the host cell's receptor through RBD. Studies have shown that RBD is a neutralizing antibody epitope of the S protein.

1-1 preparing RBD-Linker-CTB fusion gene. The RBD-Linker-CTB fusion gene comprises a codon-optimized novel coronavirus (SARS-CoV-2) RBD gene, a Linker and a CTB gene. Wherein, the original sequence of RBD gene of the novel coronavirus (SARS-CoV-2) is from NCBI, and is subjected to codon optimization to improve the expression quantity of target antigen, and the specific sequence is shown as SEQ ID NO: 1 is shown. Wherein, the gene of Linker is shown as SEQ ID NO: 2, respectively. Wherein, CTB is a B subunit of cholera toxin, and the specific sequence is shown in SEQ ID NO: 3, respectively.

In a preferred embodiment, a sequence of cleavage sites is added on both sides of the fusion gene for linking the fusion gene to a cloning vector and an expression vector.

the specific steps are that the T carrier and the RBD-Linker-CTB fusion gene are subjected to double enzyme digestion respectively, the enzyme digestion reaction system is shown in table 1, after the enzyme digestion reaction is finished, a glue recovery kit is used for recovering target fragments, then T4 DNA ligase is used for connecting the linearized RBD-Linker-CTB fusion gene and the T carrier, the reaction system is shown in table 2, the RBD-Linker-CTB fusion gene-T carrier is obtained, after the connection is finished, escherichia coli DH5 α is transformed, an LB solid resistance plate containing AMP is coated, the mixture is cultured at 37 ℃ overnight, and a positive clone is selected to be connected with an LB liquid culture medium containing AMP to extract plasmids.

1-3, connecting the RBD-Linker-CTB fusion gene to pET9a (expression vector), wherein pET9a (expression vector) and RBD-Linker-CTB fusion gene-T vector are subjected to double enzyme digestion respectively, the enzyme digestion reaction system is shown in Table 1, after the enzyme digestion reaction is finished, a glue recovery kit is used for recovering a target fragment, then T4 DNA ligase is used for connecting the linearized RBD-Linker-CTB fusion gene and pET9a to obtain the RBD-Linker-CTB fusion gene-pET 9a expression vector, the reaction system is shown in Table 2, after the connection is finished, Escherichia coli DH5 α is transformed, an LB solid resistant plate containing AMP is coated, the culture is carried out at 37 ℃ overnight, a positive clone is selected and connected with an LB liquid culture medium containing AMP, and plasmids are extracted.

1-4, double enzyme digestion identification of recombinant plasmids. Wherein, the reaction system of double enzyme cutting is shown in table 1, the double enzyme cutting result of the RBD-Linker-CTB fusion gene-T vector is shown in figure 2, HindIII and KpnI enzyme cutting sites are introduced at two sides of the RBD-Linker-CTB fusion antigen.

Table 1: double enzyme digestion reaction system (50. mu.l)

The double enzyme digestion reaction is carried out at 37 ℃ for at least more than 4 h.

TABLE 2 enzyme ligation reaction System (10. mu.l)

The ligation reaction was performed at 4 ℃ overnight.

EXAMPLE 2 construction of RBD-Linker-CRM197(A) fusion Gene of novel coronavirus SARS-CoV-2

Specific construction method referring to example 1, RBD-Linker-CRM197(A) fusion genes include codon-optimized novel coronavirus (SARS-CoV-2) RBD gene, Linker and CRM197(A) gene.

Wherein, the original sequence of the novel coronavirus (SARS-CoV-2) RBD gene is from NCBI and can improve the expression quantity of a target antigen through codon optimization, and the specific sequence is shown as SEQ ID NO: 1 is shown. Wherein the gene sequence of the Linker is GGGGS, and is shown as SEQ ID NO: 2, respectively. Wherein CRM197(A) is an A region of CRM197, and the specific sequence is shown as SEQ ID NO: shown at 9.

In the following examples, the fusion genes prepared in examples 1 and 2 are collectively referred to as RBD fusion gene of novel coronavirus (SARS-CoV-2), and simply referred to as RBD fusion gene.

Example 3 development of RBD-Linker-CTB fusion protein subunit vaccine

Transforming an RBD-Linker-CTB fusion gene-pET 9a expression vector into escherichia coli BL21, selecting a monoclonal, recovering the monoclonal in an LB liquid culture medium containing AMP, inoculating 100ml of LB liquid culture medium containing AMP for amplification until OD is reached₄₅₀Expression was induced by addition of 0.1mM IPTG (or between 0.4 and 0.6). The results of induced expression are shown in FIG. 3.

Example 4 development of RBD-Linker-CRM197(A) fusion protein subunit vaccine

With specific reference to example 3, the RBD-Linker-CRM197(A) -pET9a expression vector was transformed into E.coli BL21, and expression was induced.

Example 5 preparation of RBD-Linker-CTB fusion protein Adenoviral vector vaccine

The flow chart for packaging using the Admax adenovirus vector system is shown in FIG. 4. The 293 cells were infected with the successfully packaged adenovirus vector vaccine at MOI 5-10, and after at least 40h of infection, 8000g of the cells were centrifuged for 10min, and the cell pellet was collected. Cell pellet with PB or lysis (2mM MgCl)₂50mM HEPES, pH7.5) buffer solution, repeatedly freezing and thawing the cells at-80 ℃ for three times to lyse the cells, and centrifuging to remove cell debris.

And (3) performing ultrafiltration concentration on the supernatant by a 50KD membrane pack, passing the concentrated solution through CL-4B, collecting eluate with the volume of external water to obtain the target antigen, and performing ultrafiltration liquid exchange on the hollow fibers to obtain target buffer.

The purity of the harvested adenovirus vector vaccine was analyzed by HPLC, and a column of TSK5000 was selected, wherein the HPLC results are shown in FIG. 5, and the HPLC results show that the purity of the collected virus solution is high.

Example 6 preparation of RBD-Linker-CRM197(A) fusion protein Adenoviral vector vaccine

With specific reference to example 5, an RBD-Linker-CRM197(A) fusion protein adenoviral vector vaccine was prepared.

Example 7 preparation of mRNA of RBD-Linker-CTB fusion protein

transforming plasmid containing RBD-Linker-CTB fusion gene into Escherichia coli DH5 α, selecting positive single spot, inoculating liquid culture medium, enlarging strain, culturing bacteria in fermentation tank, centrifuging to recover bacteria, extracting plasmid, single enzyme digestion to linearize plasmid, and using the plasmid as template to synthesize mRNA.

The synthesized mRNA was capped and methylated at the front and polyA at the tail. And encapsulated in liposomes. The results of the core parameter assay before and after liposome encapsulation are as follows:

TABLE 3mRNA and Liposome encapsulation stock solution test results

The results show that the synthesized mRNA and the wrapped stock solution both meet the preparation requirements of the vaccine.

Example 8 preparation of mRNA of RBD-Linker-CRM197(A) fusion protein

With specific reference to example 7, RBD-Linker-CRM197(A) fusion protein mRNA was prepared.

Example 9 study of immunogenicity of RBD-Linker-CTB fusion proteins

And (3) taking a mouse as a model to research the immunogenicity of the RBD-Linker-CTB fusion protein. The method comprises the following specific steps: RBD-Linker-CTB fusion protein subunit vaccines, RBD-Linker-CTB fusion protein adenovirus vector vaccines and RBD-Linker-CTB fusion protein mRNA vaccines are prepared respectively for immunogenicity research.

Experimental groups: RBD-Linker-CTB fusion protein subunit vaccine

RBD-Linker-CTB fusion protein adenovirus vector vaccine

RBD-Linker-CTB fusion protein mRNA vaccine

Negative control group: physiological saline solution immunization group

Experimental animals: taking NIH mice, each group comprises 10 mice, and the weight is 12-14g

The immunization mode comprises the following steps: intramuscular injection, Single needle immunization

ImmunizationDosage: fusion protein immunization of 5 mug/mouse, and vector vaccine immunization of 1X 10⁸IFU, mRNA immunization 20. mu.g/mouse.

The serum antibody level after one-needle immunization was measured by ELISA method, and the results are shown in FIG. 6.

Experimental results show that the RBD-Linker-CTB fusion protein subunit vaccine, the RBD-Linker-CTB fusion protein adenovirus vector vaccine and the RBD-Linker-CTB fusion protein mRNA vaccine can generate good antibody level after single-needle immunization.

Example 10 investigation of immunogenicity of RBD-Linker-CRM197(A) fusion proteins

With specific reference to example 9, a study of the immunogenicity of the RBD-Linker-CRM197(A) fusion protein was performed. Experimental results show that the RBD-Linker-CRM197(A) fusion protein subunit vaccine, the RBD-Linker-CRM197(A) fusion protein adenovirus vector vaccine and the RBD-Linker-CRM197(A) fusion protein mRNA vaccine can generate good antibody level after single-needle immunization.

Example 11 Effect of molecular adjuvants TLR on immunogenicity of RBD-Linker-CTB fusion proteins

And (3) preparing an RBD-Linker-CTB fusion protein subunit vaccine containing a molecular adjuvant, an RBD-Linker-CTB fusion protein adenovirus vector vaccine and an RBD-Linker-CTB fusion protein mRNA vaccine for researching immunogenicity. To study the effect of molecular adjuvants on immunogenicity.

Experimental groups: RBD-Linker-CTB fusion protein subunit vaccine (molecular adjuvant TLR)

RBD-Linker-CTB fusion protein adenovirus vector vaccine (molecular adjuvant TLR)

RBD-Linker-CTB fusion protein mRNA vaccine (molecular adjuvant TLR)

Control group: RBD-Linker-CTB fusion protein subunit vaccine

RBD-Linker-CTB fusion protein adenovirus vector vaccine

RBD-Linker-CTB fusion protein mRNA vaccine

Negative control group: physiological saline solution immunization group

Immunization dose: fusion protein immunization of 5 mug/mouse, and vector vaccine immunization of 1X 10⁸IFU, mRNA immunization 20. mu.g/mouse.

The ELISA method was used to detect the serum antibody level after one-needle immunization, and the results are shown in FIG. 7.

Results show that the molecular adjuvant TLR can obviously improve the antibody levels of RBD-Linker-CTB fusion protein subunit vaccines, RBD-Linker-CTB fusion protein adenovirus vector vaccines and RBD-Linker-CTB fusion protein mRNA vaccines.

The same method is adopted in the embodiment, and the effect of the molecular adjuvant CPG is verified. Results show that the molecular adjuvant CPG can obviously improve the antibody levels of RBD-Linker-CTB fusion protein subunit vaccines, RBD-Linker-CTB combined protein adenovirus vector vaccines and RBD-Linker-CTB fusion protein mRNA vaccines.

Example 12 Effect of molecular adjuvants TLR on immunogenicity of RBD-Linker-CRM197(A) fusion proteins

Specifically, with reference to example 11, an experimental group, a control group, and a negative control group were prepared, respectively, and the influence of TLR, a molecular adjuvant, on the immunogenicity of RBD-Linker-CRM197(a) fusion protein was studied.

Results show that the molecular adjuvant TLR can obviously improve the antibody levels of RBD-Linker-CRM197(A) fusion protein subunit vaccine, RBD-Linker-CRM197(A) fusion protein adenovirus vector vaccine and RBD-Linker-CRM197(A) fusion protein mRNA vaccine.

The same method is adopted in the embodiment, and the effect of the molecular adjuvant CPG is verified. Results show that the molecular adjuvant CPG can obviously improve the antibody levels of RBD-Linker-CRM197(A) fusion protein subunit vaccine, RBD-Linker-CRM197(A) fusion protein adenovirus vector vaccine and RBD-Linker-CRM197(A) fusion protein mRNA vaccine.

TABLE 4 sequence composition of RBD fusion proteins

In conclusion, it was experimentally verified that the vaccines prepared in examples 1-8 can be used for the treatment and/or prevention of a novel coronavirus (SARS-CoV-2) infection or a novel coronavirus disease (COVID-19).

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

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attactttta agaatggtgc aacttttcaa gtagaagtac caggtagtca acatatagat 240

tcacaaaaaa aagcgattga aaggatgaag gataccctga ggattgcata tcttactgaa 300

gctaaagtcg aaaagttatg tgtatggaat aataaaacgc ctcatgcgat tgccgcaatt 360

agtatggcaa attaa 375

<210>4

<211>570

<212>DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

atgagcagaa aactgtttgc gtcaatctta ataggggcgc tactggggat aggggcccca 60

ccttcagccc atgcaggcgc tgatgatgtt gttgattctt ctaaatcttt tgtgatggaa 120

aacttttctt cgtaccacgg gactaaacct ggttatgtag attccattca aaaaggtata 180

caaaagccaa aatctggtac acaaggaaat tatgacgatg attggaaagg gttttatagt 240

accgacaata aatacgacgc tgcgggatac tctgtagata atgaaaaccc gctctctgga 300

aaagctggag gcgtggtcaa agtgacgtat ccaggactga cgaaggttct cgcactaaaa 360

gtggataatg ccgaaactat taagaaagag ttaggtttaa gtctcactga accgttgatg 420

gagcaagtcg gaacggaaga gtttatcaaa aggttcggtg atggtgcttc gcgtgtagtg 480

ctcagccttc ccttcgctga ggggagttct agcgttgaat atattaataa ctgggaacag 540

gcgaaagcgt taagcgtaga acttgagatt 570

<210>5

<211>69

<212>DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60

tcgcccagc 69

<210>6

<211>224

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>6

Met Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr

1 5 10 15

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser

20 25 30

Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr

35 40 45

Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly

50 55 60

Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala

65 70 75 80

Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly

85 90 95

Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

100 105 110

Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val

115 120 125

Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu

130 135 140

Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser

145 150 155 160

Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln

165 170 175

Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg

180 185 190

Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys

195 200 205

Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe

210 215 220

<210>7

<211>5

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>7

Gly Gly Gly Gly Ser

1 5

<210>8

<211>124

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>8

Met Ile Lys Leu Lys Phe Gly Val Phe Phe Thr Val Leu Leu Ser Ser

1 5 10 15

Ala Tyr Ala His Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu

20 25 30

Tyr His Asn Thr Gln Ile His Thr Leu Asn Asp Lys Ile Phe Ser Tyr

35 40 45

Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys

50 55 60

Asn Gly Ala Thr Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp

65 70 75 80

Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala

85 90 95

Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys

100 105 110

Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn

115 120

<210>9

<211>188

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>9

Met Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu

1 5 10 15

Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile

20 25 30

Gln Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp

35 40 45

Asp Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala

50 55 60

Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly

65 70 75 80

Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys

85 90 95

Val Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr

100 105 110

Glu Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe

115 120 125

Gly Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly

130 135 140

Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu

145 150 155160

Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln

165 170 175

Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala

180 185

Claims

1. An RBD fusion protein comprising:

(1) RBD amino acid fragments of novel coronavirus SARS-CoV-2;

(2) a Linker is connected with the arm;

(3) CTB or CRM197(A) amino acid fragment.

2. The fusion protein of claim 1,

(2) the sequence of the connecting arm Linker is shown as SEQ ID NO: 7, and/or,

3. The fusion protein of claim 1 or 2, further comprising: a signal peptide and/or a molecular adjuvant; preferably, the signal peptide is connected to the N end of the fusion protein, and the molecular adjuvant is connected to the C end of the fusion protein; more preferably, the signal peptide is represented by the sequence shown in SEQ ID NO: and 7, connecting a Linker arm Linker shown in the figure and the fusion protein.

4. The fusion protein of claim 3,

(1) the signal peptide is tPA signal peptide, and/or,

(2) the molecular adjuvant is CPG or TLR agonist.

5. A fusion gene characterized by comprising a gene encoding a polypeptide,

(1) encoding the fusion protein of any one of claims 1-4;

alternatively, the first and second electrodes may be,

6. A vector comprising the fusion gene of claim 5.

7. A host expressing the fusion protein of any one of claims 1 to 4, comprising the fusion gene of claim 5 and/or comprising the vector of claim 6.

8. A vaccine for treating and/or preventing a novel coronavirus SARS-CoV-2 infection or a novel coronavirus disease COVID-19, comprising the fusion protein according to any one of claims 1 to 4, the fusion gene according to claim 5, the vector according to claim 6, the host according to claim 7, or produced from the fusion protein according to any one of claims 1 to 4, the fusion gene according to claim 5, the vector according to claim 6, or the host according to claim 7.

9. The vaccine of claim 8, wherein said vaccine is in the form of a recombinant protein subunit vaccine, a recombinant protein mRNA vaccine, or a recombinant protein adenoviral vector vaccine.

10. A preparation method of a vaccine for preventing and/or treating novel coronavirus SARS-CoV-2 infection or coronavirus disease COVID-19 is characterized by comprising the following steps:

(1) constructing the fusion gene according to claim 5,

(2) expressing the fusion protein of any one of claims 1 to 4,

(3) purifying to obtain subunit vaccine;

alternatively, the first and second electrodes may be,

(1) constructing the fusion gene according to claim 5,

(2) inserting the fusion gene into an adenovirus vector,

(3) preparing an adenovirus vector vaccine;

alternatively, the first and second electrodes may be,

(1) the fusion gene of claim 5 as a template,

(2) and amplifying to obtain the mRNA vaccine.

11. Use of the fusion protein according to any one of claims 1 to 4, the fusion gene according to claim 5, the vector according to claim 6, the host according to claim 7 and/or the vaccine according to any one of claims 8 to 9 for the preparation of a medicament or product for the prevention and/or treatment of a novel coronavirus SARS-CoV-2 infection and/or a novel coronavirus disease covi-19.

12. A method for preventing and/or treating a novel coronavirus SARS-CoV-2 infection and/or a novel coronavirus disease CoV-19, comprising administering the fusion protein of any one of claims 1 to 4, the fusion gene of claim 5, the vector of claim 6, the host of claim 7, and/or the vaccine of any one of claims 8 to 9 to an individual.

13. A method of inducing a neutralizing antigen-specific immune response in an individual, characterized in that a fusion protein according to any one of claims 1 to 4, a fusion gene according to claim 5, a vector according to claim 6, a host according to claim 7 and/or a vaccine according to any one of claims 8 to 9 is administered to the individual.